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Molecular Endocrinology, doi:10.1210/me.2005-0360
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Molecular Endocrinology 20 (3): 661-674
Copyright © 2006 by The Endocrine Society

Two Wrongs Can Make a Right: Dimers of Prolactin and Growth Hormone Receptor Antagonists Behave as Agonists

John F. Langenheim, Dunyong Tan, Ameae M. Walker and Wen Y. Chen

Department of Biological Sciences (J.F.L., W.Y.C.), Clemson University, Clemson, South Carolina 29634-0326; Oncology Research Institute (J.F.L, W.Y.C), Greenville Hospital System, Greenville, South Carolina 29605-4255; and Division of Biomedical Sciences (D.T, A.M.W.), University of California, Riverside, Riverside, California 92521-0121

Address all correspondence and requests for reprints to: Wen Y. Chen, Oncology Research Institute, Greenville Hospital System, 900 West Faris Road, Greenville, South Carolina 29605-4255. E-mail: wchen{at}ghs.org.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Prolactin (PRL) and GH have two distinct binding sites (site 1 with high affinity; site 2 with low affinity) that each interact with a PRL receptor (PRLR) to form a functional receptor dimer that activates signal transduction. The G129R mutation in PRL and the G120R mutation in GH disrupt the structural integrity of site 2 such that the ligands retain the ability to bind to the first receptor with high affinity, but act as receptor antagonists. In this study, we examined the ability of monomeric and dimeric forms of these ligands, human (h) PRL and hGH, and their antagonists (hPRL-G129R and hGH-G120R) to 1) bind to PRLRs; 2) induce conformational changes in PRLRs; 3) activate signaling pathways associated with the PRLR; and 4) mediate cell proliferation in vitro. In contrast to monomeric hPRL-G129R, homodimeric hPRL-G129R induced PRLR dimerization; activated Janus family of tyrosine kinases 2/signal transducer and activator of transcription 5, Ras/Raf/MAPK kinase/Erk, and phosphatidylinositol 3-kinase/Akt signaling; and stimulated Nb2 cell proliferation. Similarly, homodimeric hGH-G120R was able to mediate signaling via the PRLR and to stimulate Nb2 cell proliferation. These experiments demonstrate that a ligand must have two functional binding sites, but that these may be site 1 plus site 2 or two site 1’s, to elicit receptor-mediated signal transduction. The size of the ligand plays less of a role in receptor activation, suggesting that the extracellular portion of the PRLR (and possibly the GH receptor) is rather flexible and can accommodate larger ligands. These findings may have implications for designing multifunctional therapeutics that target this class of cytokine receptors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PROLACTIN (PRL) AND GH are members of the hematopoietin family of cytokines. Despite their low amino acid sequence identity, they have similar tertiary structures (1, 2), as do the extracellular regions of the receptors to which they bind (3). These peptide hormones have four {alpha}-helical segments that are connected via loops that provide flexibility and allow the helices to be bundled in an antiparallel (up-up-down-down) manner. Two separate asymmetric receptor-binding regions have been identified in these hormones, each of which interacts with the equivalent region of a receptor to form a one-ligand, two-receptor complex. Due to differences in affinity between the two receptor-binding sites, binding occurs sequentially with the higher affinity site (site 1) interacting with the first receptor before the lower affinity site (site 2) can interact with a second receptor (4, 5, 6, 7, 8). Recently, this sequential binding process has been shown to be due to the functional coupling of the receptor-binding sites. The interaction of site 1 with the first receptor induces conformational changes in the ligand that create a functional site 2 (9, 10).

Studies of GH, PRL, and placental lactogen (PL) have shown that the third {alpha}-helix is important to their structure and the function of site 2. Substitution of a Gly residue in this region with an Arg residue results in antagonists (hGH-G120R, hPRL-G129R, hPL-G120R) with little or no biological activity (6, 8, 11, 12, 13, 14). Comparative studies of the interaction of hGH-G120R and hGH with the extracellular domain (ECD) of the human GH receptor (hGHR-ECD) have shown that the mutation has minimal effects upon initial hormone-receptor interactions at site 1 but disrupts the critical hormone-receptor-interactions at site 2 (1, 15, 16), thereby preventing proper receptor dimerization.

Proper ligand-induced receptor dimerization induces conformational changes in the receptors that bring about the activation of receptor-associated Janus family of tyrosine kinases 2 (JAK2) (17, 18, 19, 20, 21), which stimulates signal transducer and activators of transcription 5 (STAT5) signaling. Receptor activation also leads to the activation of Ras/Raf/MAPK kinase/Erk (22, 23) and phosphatidylinositol 3-kinase/Akt signaling pathways. It is primarily via these pathways that the receptors for these ligands induce cell differentiation, proliferation, and/or survival (24, 25).

In addition to the monomeric forms of these ligands, dimeric and oligomeric forms have been identified in plasma of mammals and within pituitary extracts (26, 27, 28, 29, 30). Dimeric forms may arise from interchain disulfide linkages or noncovalent aggregation; oligomeric forms exist and larger forms may also arise from IgG covalent and noncovalent complexes with monomeric or dimeric forms of these ligands (29, 31, 32, 33). The significance of these larger forms with reduced affinities for PRLRs remains largely unknown. In addition to these natural ligands of the PRL receptor (PRLR), bivalent monoclonal antibodies (mAbs) have been developed that exhibit various degrees of agonistic activity (34, 35, 36) that correlate with their ability to induce the phosphorylation of JAK2 (37).

To better understand the molecular interactions between ligands and the PRLR, we prepared dimeric forms of hGH, hPRL, and their antagonists including homodimers (PRL-PRL, GH-GH) with four potentially functional binding sites, heterodimers (PRL-G129R, G129R-PRL) with three potential binding sites, and homodimers (G129R-G129R, G120R-G120R) with two potentially functional binding sites. We demonstrate that the fusion of two antagonists (hPRL-G129R or hGH-G120R) together restores some biological activity, even though the two functional binding sites are located in separate moieties. This suggests that the PRL/GH receptor family exhibits a considerable degree of flexibility in accommodating ligands and transducing their signals. This finding may have implications for designing ligand-based therapeutics that target this family of receptors.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Construction, Expression, and Purification of Dimeric Derivatives of hPRL and hGH
Homodimers and heterodimers of hPRL and hPRL-G129R were recombinantly engineered by gene fusion. The cDNAs encoding the mature form of the proteins were ligated together via a small linker (GGATCC) and cloned into an Escherichia coli expression vector. Expression of the fusion gene in E. coli resulted in a single-fusion protein in which the carboxy terminus of the first moiety was connected to the amino terminus of the second moiety by a Gly-Ser peptide linker. The proteins were found to accumulate in the insoluble fraction as inclusion bodies. The inclusion bodies were isolated, denatured, refolded, and purified by anion-exchange chromatography to yield preparations of greater than 95% purity. The dimeric fusion proteins were analyzed by nonreducing (Fig. 1AGo) and reducing SDS-PAGE (Fig. 1BGo) along with hPRL and hPRL-G129R. hPRL and hPRL-G129R were predominantly monomeric (20 kDa) under nonreducing conditions with only a small portion remaining unfolded (25 kDa) or forming covalently linked dimers via interchain disulfide linkages (40 kDa) (Fig. 1AGo, lanes 1 and 2). No covalently linked multimeric forms were observed for the recombinantly engineered homodimers and heterodimers of hPRL and hPRL-G129R after purification; however, multiple bands were observed, which would suggest some improper intrachain disulfide linkages (Fig. 1AGo, lanes 3–6). This was anticipated because the carboxy-terminal cysteines of the first moiety (Cys191, Cys199) are separated from the amino-terminal cysteines (Cys4, Cys11) of the second moiety by only a small linker (Gly-Ser). Reducing SDS-PAGE revealed that purified dimers have sizes corresponding to their predicted molecular mass of 46 kDa (Fig. 1BGo, lanes 3–6), and Western blotting with an antibody that detects hPRL and hPRL-G129R confirmed the identity of purified dimers (Fig. 1CGo, lanes 3–6).


Figure 1
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  		Fig. 1. Nonreducing SDS-PAGE, Reducing SDS-PAGE, and Western Blot Analysis of Purified Monomeric and Dimeric hPRL Derivatives

Monomers, homodimers, and heterodimers of hPRL and hPRL-G129R were separated by nonreducing SDS-PAGE (A) or reducing SDS-PAGE (B) on 12% polyacrylamide gels and stained with SYPRO Orange to confirm their size and purity. M, BenchMark Ladder (Invitrogen); lane 1, PRL; lane 2, G129R; lane 3, PRL-PRL; lane 4, G129R-G129R; lane 5, PRL-G129R; and lane 6, G129R-PRL. C, The identity of the proteins was confirmed by Western blotting with an antibody that detects the hPRL and hPRL-G129R moieties.

 
Homodimers of hPRL and hPRL-G129R Retain the Ability to Bind to hPRLRs
To examine whether the homodimers of hPRL and hPRL-G129R retain the ability to bind to PRLRs, we measured their ability to compete with 125I-labeled hPRL for binding to PRLRs expressed on the surface of T-47D human breast cancer cells. Monomers and homodimers of hPRL and hPRL-G129R effectively displaced the binding of [125I]hPRL (Fig. 2Go), confirming that the dimers retain the ability to specifically bind to hPRLRs. The effective concentrations necessary to displace 50% of the 125I-labeled hPRL (EC50) were calculated. There was no statistical difference between the EC50 of dimeric hPRL (0.97 ± 0.23 nM), dimeric hPRL-G129R (1.17 ± 0.1 nM), and monomeric hPRL (0.96 ± 0.29 nM); however, there were statistical differences between the EC50 of these ligands and monomeric hPRL-G129R (1.96 ± 0.25 nM).


Figure 2
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  		Fig. 2. Competitive Binding of Monomeric and Homodimeric hPRL Derivatives to hPRLRs on T-47D Cells

The ability of monomeric and homodimeric hPRL and hPRL-G129R to bind to hPRLRs was determined by measuring their ability to compete with 125I-labeled hPRL for binding to the surface of T-47D cells as described in Materials and Methods. Each data point represents the mean ± SD of at least three independent experiments. The EC50 values for monomeric hPRL, homodimeric hPRL, homodimeric hPRL-G129R, and monomeric hPRL-G129R were determined to be 0.96 ± 0.29 nM, 0.97 ± 0.23 nM, 1.17 ± 0.1 nM, and 1.96 ± 0.25 nM, respectively. *, P < 0.05; **, P < 0.005, for difference in EC50 from that of monomeric hPRL.

 
Homodimeric hPRL-G129R Induces a Bioluminescence Resonance Energy Transfer (BRET) Signal Consistent with Receptor Dimerization
To determine whether dimeric hPRL-G129R truly has a second functional binding site, we examined whether or not it could induce conformational changes in hPRLRs consistent with dimerization. Figure 3AGo shows representative luminescence scans in the absence or presence of monomeric hPRL, monomeric hPRL-G129R, and homodimeric hPRL-G129R. In the absence of ligand or the presence of monomeric hPRL-G129R, there is minimal emission of a BRET signal from human embryonic kidney (HEK) 293 cells cotransfected with tagged hPRLRs, SF1b-Renilla reniformis luciferase (Rluc)/SF1b-green fluorescent protein (GFP2), whereas, the addition of monomeric hPRL or homodimeric hPRL-G129R induces a significant BRET signal (Fig. 3AGo). The BRET ratios for HEK 293 cells cotransfected with long form (LF)-Rluc/LF-GFP2, short form (SF)1a-Rluc/SF1a-GFP2, or SF1b-Rluc/SF1b-GFP2 increased approximately 4-, 3-, and 3-fold, respectively, upon treatment with dimeric hPRL-G129R (Fig. 3BGo).


Figure 3
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  		Fig. 3. Monomeric hPRL and Homodimeric hPRL-G129R-Induced Homodimerization of hPRLRs

BRET2 assays were performed within 48 h after transfection of HEK 293 cells in the presence of 5 µM DeepBlueC (DBC) with or without monomeric hPRL, monomeric hPRL-G129R, or homodimeric hPRL-G129R as described in Materials and Methods. A, Representative bioluminescence scans from the DBC reaction in the absence of ligand and after the addition of monomeric hPRL (45.7 nM), monomeric hPRL-G129R (45.7 nM), or homodimeric hPRL-G129R (45.7 nM) to cells transiently transfected with SF1a-GFP2/SF1a-Rluc. Note the absence of a BRET signal in the absence of monomeric hPRL or homodimeric hPRL-G129R. B, Averaged BRET ratios from four or more separate determinations on different preparations of cells transfected with LF, SF1a, or SF1b. The BRET ratio was calculated as (Emission500–520 nm – Background500–520 nm)/(Emission385–420 nm – Background385–420 nm).*, P < 0.05; **, P < 0.01, for difference with and without monomeric hPRL or dimeric hPRL-G129R.

 
Dimerization of hPRLR Antagonists Restores hPRLR-Mediated Signal Transduction Pathways
To determine whether the receptor dimers induced by homodimeric hPRL-G129R are functional, we examined its ability to activate signaling molecules associated with the PRLR. We also produced homodimeric hGH-G120R, a GHR as well as PRLR antagonist, to test whether dimerization of this antagonist would also produce an agonist. Treatment of T-47D cells with various concentrations of hPRL or hGH resulted in the phosphorylation of STAT5, Erk1/2, and Akt (Fig. 4AGo). Monomeric hPRL-G129R and hGH-G120R, which block the conformational changes necessary for the formation of a functional receptor dimer, did not elicit any of these signaling molecules (Fig. 4AGo). In contrast to the monomeric hPRLR antagonists, homodimers of hPRL-G129R and hGH-G120R activated STAT5, Erk1/2, and Akt in a dose-dependent manner (Fig. 4BGo).


Figure 4
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  		Fig. 4. Western Blot Analysis of the Phosphorylation Status of Akt, Erk1/2, and STAT5A/B in Response to Monomeric and Dimeric hPRL and hGH Derivatives

T-47D cells were plated as described in Materials and Methods and serum starved overnight. The cells were treated for 30 min with 5 nM, 50 nM, or 500 nM of: (A) monomeric hPRL, hPRL-G129R, hGH or hGH-G120R; (B) homodimeric hPRL, hPRL-G129R, hGH or hGH-G120R; or (C) heterodimers of hPRL and hPRL-G129R. Cell lysates were harvested, and 40 µg of lysate from each treatment was separated by reducing SDS-PAGE using 4–15% polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with antibodies recognizing phospho-Akt1/2/3, phospho-ERK1/2, and phospho-STAT5A/B. Equal loading was confirmed by reprobing for STAT5. P-, Phospho.

 
Dose response and time course experiments were performed to examine the potency (Fig. 5Go) and efficiency (see Fig. 7Go) of dimeric hPRL-G129R in the activation of STAT5 in comparison with hPRL. Dimeric hPRL-G129R was able to induce STAT5 phosphorylation (Fig. 5BGo) but required approximately 3-fold higher molar concentrations to achieve the same degree of STAT5 phosphorylation observed for hPRL (Fig. 5AGo). The difference in potency between dimeric hPRL-G129R and monomeric hPRL may be explained, in part, by the difference in their relative binding affinity to hPRLRs (Fig. 2Go) but may also be related to the adjustments in the receptor that are necessary to bind two site 1’s. We also compared the efficiency of dimeric hGH-G120R and monomeric hGH at activating STAT5, Erk1/2, and Akt (Fig. 6Go). Dimeric hGH-G120R induced the phosphorylation of these signaling molecules in a dose-dependent manner (Fig. 6BGo) but required approximately 30-fold higher concentrations to achieve similar levels of activation induced by monomeric hGH (Fig. 6AGo).


Figure 5
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  		Fig. 5. Dose-Dependent Activation of STAT5A/B Phosphorylation Induced by Monomeric and Dimeric hPRL Derivatives

T-47D cells were plated as described in Materials and Methods and serum starved overnight. The cells were treated with increasing doses of monomeric (A), homodimeric (B and C), or heterodimeric (D and E) hPRL or hPRL-G129R, and the efficiency of STAT5A/B phosphorylation was examined after 20 min. Cell lysates were harvested and 40 µg of lysate from each treatment was separated by reducing SDS-PAGE using 4–15% polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with anti-phospho-STAT5A/B. Equal loading was confirmed by reprobing the membranes with an anti-STAT5 antibody. P-Stat5, Phospho-Stat5.

 

Figure 7
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  		Fig. 7. Time Course of Erk1/2 and STAT5A/B Phosphorylation in T-47D Cells in Response to Monomeric and Dimeric hPRL Derivatives

T-47D cells were plated as described in Materials and Methods and serum starved overnight. The cells were treated with 10 nM of monomeric hPRL (A), homodimeric hPRL-G129R (B), homodimeric hPRL (C), or heterodimeric hPRL and hPRL-G129R (D and E) for various durations (0, 2, 5, 10, 20, 30, 40, 50, and 60 min). Cell lysates were harvested, and 40 µg of lysate from each treatment was separated by reducing SDS-PAGE using 4–15% polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with antibodies that recognize phospho-Erk1/2 and phospho-STAT5A/B. Equal loading was confirmed by reprobing the membranes with an anti-STAT5 antibody. P-, Phospho.

 

Figure 6
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  		Fig. 6. Dose-Dependent Induction of STAT5A/B, Erk1/2, and Akt Phosphorylation Induced by Monomeric and Dimeric hGH Derivatives

T-47D cells were plated as described in Materials and Methods and serum starved overnight. The cells were treated with increasing doses of monomeric hGH (A), homodimeric hGH-G120R (B), or homodimeric hGH (C), and the efficiency of activation was examined after 30 min. Cell lysates were harvested and 40 µg of lysate from each treatment was separated by reducing SDS-PAGE using 10% polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with antibodies that recognize phospho-STAT5A/B, phospho-ERK1/2, and phospho-Akt1/2/3. Equal loading was confirmed by reprobing the membranes with an anti-STAT5 antibody. P-, Phospho.

 
Consistent with having a lower affinity, the efficiency of signal transduction induced by 10 nM dimeric hPRL-G129R (Fig. 7BGo) or dimeric hGH-G120R (Fig. 8BGo) were lower than that of 10 nM of hPRL (Fig. 7AGo) and hGH (Fig. 8AGo), requiring a longer time course to achieve similar activation levels. The phosphorylation of STAT5 induced by 10 nM dimeric hPRL-G129R (Fig. 7AGo) was detectable after 20 min, whereas it was detectable after 10 min for hPRL (Fig. 7BGo). The phosphorylation of STAT5 by dimeric hGH-G120R (Fig. 8BGo) required 30 min to achieve the same level as hGH (Fig. 8AGo) after 2 min; similarly, it took much longer for dimeric hGH-G120R to stimulate phosphorylation of Erk1/2.


Figure 8
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  		Fig. 8. Time Course of STAT5A/B, Akt, and ERK1/2 Phosphorylation in T-47D Cells in Response to Monomeric and Dimeric hGH Derivatives

T-47D cells were plated as described in Materials and Methods and serum starved overnight. The cells were treated with 10 nM of monomeric hGH (A), homodimeric hGH-G120R (B), or homodimeric hGH (C) for various durations (0, 2, 5, 10, 20, 30, 40, 50, and 60 min). Cell lysates were harvested, and 40 µg of lysate from each treatment was separated by reducing SDS-PAGE using 10% polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with antibodies that recognize phospho-STAT5A/B, phospho-Akt1/2/3, and phospho-Erk1/2. Equal loading was confirmed by reprobing the membranes with an anti-STAT5 antibody. P-, Phospho.

 
We further examined the signaling induced by homodimers of hPRL, homodimers of hGH, and heterodimers of hPRL and hPRL-G129R. We show that these dimers activate the same signaling pathways as hPRL and hGH (Fig. 4Go). Heterodimers of hPRL and hPRL-G129R (Fig. 5Go, D and E, and Fig. 7Go, D and E) exhibited slightly lower signaling efficiencies than monomeric hPRL (Figs. 5AGo and 7AGo), whereas homodimers of hPRL (Figs. 5CGo and 7CGo) and hGH (Figs. 6CGo and 8CGo) activated STAT5 nearly as well as monomeric hPRL (Figs. 5AGo and 7AGo) and hGH (Figs. 6AGo and 8AGo).

Dimeric hPRL-G129R and hGH-G120R Induce Proliferation of Rat Nb2 Cells in a Dose-Dependent Manner
To examine the biological activity of homodimeric hPRL-G129R, homodimeric hGH-G120R, and the other dimeric derivatives, we measured their ability to induce the proliferation of rat Nb2 cells, which proliferate in response to lactogenic hormones. The relative number of viable cells was determined after 72 h treatment by colorimetrically measuring the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) by living cells. Treatment of the cells with increasing doses of homodimeric hPRL-G129R (Fig. 9AGo) or homodimeric hGH-G120R (Fig. 9CGo) stimulated cell proliferation in a dose-dependent manner. Homodimers of hPRL (Fig. 9BGo), homodimers of hGH (Fig. 9DGo), and heterodimers of hPRL and hPRL-G129R (Fig. 9Go, E and F) were more potent agonists than homodimers of hPRL-G129R (Fig. 9AGo) and homodimers of hGH-G120R (Fig. 9CGo). The proliferation induced by these ligands in rat Nb2 cells correlated well with the ability of the ligands to bind receptors (Fig. 2Go) and induce signaling in human T-47D cells (Figs. 4–6GoGoGo). Dimeric hPRL-G129R appeared to be a more potent agonist than dimeric hGH-G120R.


Figure 9
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  		Fig. 9. Rat Nb2 Cell Proliferation in Response to Monomeric and Dimeric hPRL and hGH Derivatives

Rat Nb2 cells were treated with various doses of monomeric, homodimeric, or heterodimeric hPRL, hPRL-G129R, hGH, or hGH-G120R for 72 h as described in Materials and Methods. Cell proliferation was assessed by colorimetrically measuring the reduction of MTS. Each data point represents the mean ± SD of at least three independent experiments performed in quadruplicate. Rat Nb2 cells proliferate in response to picomolar concentrations of hPRL and hGH; hPRL-G129R and hGH-G120R exhibit partial agonism at concentrations more than 2-log higher than that of hPRL and hGH. This partial agonism has been attributed to the rat PRLR forming a more stable interaction with binding site 2 of these ligands than the hPRLR.

 
To provide further evidence that the agonism observed for homodimeric hPRL-G129R was due to the presence of two functional site 1’s, we made a series of deletions to the carboxy terminus of the second moiety of hPRL-G129R (Fig. 10Go). Removal of a portion of helix IV by deleting the last 25 amino acid residues (G129R2{Delta}C25) reduced the agonism by approximately 10-fold. Further removal of helix IV by deleting 40 amino acid residues (G129R2{Delta}C40) resulted in further reduction in agonist activity to a level similar to monomeric hPRL-G129R, strongly suggesting that helix IV in the second moiety of hPRL-G129R plays an important role in forming the second functional binding site. Interestingly, truncations into and beyond helix III (G129R2{Delta}C80 thru G129R2{Delta}C160, with the exception of G129R2{Delta}C180) abolished all residual agonistic activity.


Figure 10
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  		Fig. 10. Examination of Rat Nb2 Cell Proliferation in Response to Truncated Forms of Dimeric hPRL-G129R

Rat Nb2 cells were treated with various doses of hPRL, hPRL-G129R, or truncated forms of dimeric hPRL-G129R (G129R2) for 72 h as described in Materials and Methods. Cell proliferation was assessed by colorimetrically measuring the reduction of MTS. Each data point represents the mean ± SD of at least two independent experiments performed in quadruplicate.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
hPRL and hGH share limited amino acid sequence identity, yet they both can bind to hPRLRs with high affinity to form functional receptor dimers. These ligands have two separate and different binding sites that each interact with a PRLR in a sequential manner to form a 1:2 ligand-receptor complex that activates signal transduction (1, 5, 6, 38, 39). Although the interactions of hPRL and hGH with the PRLR overlap, they are not identical (40): hGH requires the presence of zinc to bind with high affinity (41). The ability of hGH to adapt to both the GHR and PRLR has largely been attributed to the local conformational flexibility of its binding loops (42). Studies have shown that the second half of the long loop between helices I and II is important to the structure and/or function of both GH and PRL (43, 44). Conformational changes in this region of the loop were observed for a high-affinity hGH variant (45), and amino acid changes to this region were necessary to engineer PRL to bind to the GHR (46). These studies suggest that the natural ligands of the PRLR exhibit local conformational flexibility.

The ability of the PRLR and GHR to be activated by ligands other than the monomeric forms suggests that the ECDs of the PRLR and GHR also exhibit flexibility. In addition to monomers, dimeric and oligomeric forms of PRL, GH, and PL have been identified in the circulation of mammals and account for a small portion of the total plasma levels of these ligands (27, 28, 29, 30). Dimeric forms may arise from interchain disulfide linkages, linkages between glycosylated monomers, or be due to noncovalent interactions. Purified disulfide-linked dimers of hPL have been shown to retain the ability to bind to mammary glands with high affinity and to be biologically active in vitro (33, 47). A natural dimeric form of hGH with an extremely stable disulfide linkage (MER-45-kDA hGH) has been characterized (48), which binds to both the GHR and PRLR with high affinity (49). It has been shown to stimulate Nb2 cell proliferation (50), but to inhibit the proliferation of breast cancer cell lines (51), suggesting either that it acts as an antagonist in the presence of autocrine PRL or that it antagonizes the GHR, or both (52). In addition to these natural ligands, mAbs developed against the ECD of the PRLR have been shown to be weak agonists (34, 35, 36), with activities approximately 100-fold lower than natural monomeric ligands in the rat Nb2 assay (37). These bivalent mAbs were inactive as monovalent Fab fragments, confirming that the dimerization and activation of PRLRs occur only when a ligand has two functional binding sites. Whether or not these mAbs recognize epitopes involved in PRL binding remains to be determined.

To specifically address whether or not the ligand-binding sites of the hPRLR are flexible enough to accommodate larger ligands, we recombinantly engineered dimeric forms of the ligands (hPRL, hGH) and their antagonists (hPRL-G129R, hGH-G120R). Previous studies have shown that mutations in helix III of GH and PRL disrupt the integrity of binding site 2, resulting in mutants with little or no biological activity (6, 8, 11, 12, 13, 14). This Gly to Arg mutation reportedly has minimal effects upon initial hormone-receptor interactions at site 1 but disrupts the critical hormone-receptor interactions at site 2 (1, 15, 16), preventing proper receptor dimerization from occurring. We reasoned that the fusion of two molecules of hPRL-G129R or hGH-G120R together would create a bivalent ligand with only two potentially functional binding site 1’s located in different moieties. Dimerization and activation of PRLR by these dimers would provide further evidence that the PRLR exhibits considerable flexibility in accommodating ligands of larger size.

Using a radioreceptor binding assay we examined the overall affinity of hPRL derivatives for hPRLRs expressed on the surface of T-47D cells. Because ligand binding occurs sequentially and because ligand binding is transient in nature, the radioreceptor binding assay reflects 1:1 ligand-receptor interactions to a larger extent than 1:2 interactions. We observed that higher concentrations of hPRL-G129R were needed to compete with 125I-labeled hPRL than were necessary for hPRL (Fig. 2Go) (14). This lower affinity may reflect the inability of hPRL-G129R to interact with a second receptor due to the disruption of binding site 2. Homodimers of hPRL-G129R were found to have an affinity greater than that of monomeric hPRL-G129R (Fig. 2Go), indicating that the addition of a second hPRL-G129R moiety was able to improve the interaction of the ligand with a second receptor. Homodimeric hPRL was found to have an affinity for hPRLRs similar to that of monomeric hPRL, suggesting that the fusion of two hPRL molecules together had minimal effect on 1:1 ligand-receptor interactions.

To determine whether dimeric hPRL-G129R truly has a second functional binding site, we examined whether or not it could induce the dimerization of hPRLRs. Ligand-induced dimerization causes conformational changes that bring the cytoplasmic domains of the two PRLRs into close proximity to each other. To examine the conformational changes induced by dimerization, we fused Rluc or a mutant of green fluorescent protein (GFP2) to the carboxy terminus of the long (LF) and short (SF1a, SF1b) forms of the hPRLR and cotransfected the fusions of each isoform into HEK 293 cells. Upon addition of DeepBlueC, Rluc catalytically degrades the substrate and releases bioluminescence resonance energy (peak ~395 nm). If receptor dimerization occurs, Rluc is brought into close proximity to GFP2, and the bioluminescence resonance energy is transferred to GFP2, which emits fluorescence at a higher wavelength (peak ~ 515 nm) referred to as a BRET signal. Previously, it has been demonstrated that hPRL induces a BRET signal in this system and that hPRL-G129R does not; additionally, it has been shown that hPRL-G129R can competitively inhibit hPRL-induced BRET (53). In contrast to monomeric hPRL-G129R, we demonstrate that the addition of homodimeric hPRL-G129R to the transfected cells results in the emission of a BRET signal similar to that of monomeric hPRL (Fig. 3AGo). The BRET ratios for HEK 293 cells cotransfected with LF-Rluc/LF-GFP2, SF1a-Rluc/SF1a-GFP2, or SF1b-Rluc/SF1b-GFP2 and treated with homodimeric hPRL-G129R increased approximately 4-, 3-, and 3-fold, respectively (Fig. 3BGo); these values are comparably less than that of monomeric hPRL which increased the BRET ratios approximately 9-, 4-, and 4-fold, respectively (53). The lower efficiency of BRET induction by homodimeric hPRL-G129R in comparison with monomeric hPRL may reflect alterations in the conformation of the receptor homodimers formed. These findings indicate that dimeric hPRL-G129R is able to induce conformational changes in the long and short isoforms of the hPRLR that are consistent with receptor dimerization. It also indicates that the second hPRL-G129R moiety provides the second functional receptor-binding site necessary for receptor dimerization to occur.

To determine whether the receptor dimers induced by dimeric hPRL-G129R were functional, we examined the ability of dimeric hPRL-G129R to activate signaling molecules. The conformational changes induced by the formation of a functional PRLR dimer activate receptor-associated JAK2 tyrosine kinase, which stimulates the phosphorylation of STAT5. Activation of hPRLRs by hPRL or hGH also leads to the activation of the Ras/Raf/MAPK kinase/Erk and phosphatidylinositol 3-kinase/Akt signaling pathways. We demonstrate that dimeric hPRL-G129R and dimeric hGH-G120R are able to activate these three different intracellular signaling pathways in a manner qualitatively similar to that of hPRL and hGH (Fig. 4Go). The magnitude of response induced by the dimers was similar to that of monomeric hPRL and hGH (Figs. 5Go and 6Go); however, activation of signaling by homodimeric hPRL-G129R was slightly less efficient than hPRL, whereas homodimeric hGH-G120R was significantly less efficient than hGH. These finding suggest that dimeric hPRL-G129R acts as a substantial agonist and that hGH-G120R acts as a weak agonist at the hPRLR.

One explanation for the reduced efficiency of homodimeric hPRL-G129R and homodimeric hGH-G120R is that the receptor homodimers formed upon ligand binding have an altered conformation that reduces the efficiency of signal transduction. The even lower efficiency of signaling induced by homodimeric hGH-G120R may be attributed to the signaling of hGH and its derivatives being mediated primarily via the PRLR and not the GHR in T-47D cells (54). Studies have shown that His18 and Glu174 located within binding site 1 of hGH and Asp187 and His188 of the hPRLR interact with a single zinc cation to enhance the affinity of hGH for the hPRLR approximately 8000-fold (41, 55). Presumably, homodimeric hGH would require the coordination of two zinc cations to dimerize PRLRs (one for each binding site 1) because the two binding site 2s are not functional. This would suggest that the interactions of homodimeric hGH-G120R with PRLRs may be more stringent and less flexible than would be necessary for this ligand to interact with the GHR.

The quantitatively similar signaling activation observed for monomeric hPRL (Figs. 4AGo, 5AGo, and 7AGo) and heterodimers of hPRL and hPRL-G129R (Fig. 4CGo, Fig. 5Go, D and E, and Fig. 7Go, D and E) suggests that fusion of two ligands together has little effect on their ability to interact with the first receptor, a finding in agreement with the binding data. The decreased agonism observed for homodimeric hPRL-G129R (Figs. 4BGo, 5BGo, and 7BGo), as opposed to heterodimers, likely reflects the fact that both moieties are required for activity, requiring some accommodation in the receptors. These data indicate that the receptor dimerization induced by dimeric derivatives of hPRL and hGH are functional and capable of activating the signaling pathways necessary for biological activity.

hPRL-mediated signaling can induce cell differentiation, proliferation, and/or survival (24, 25). We examined the bioactivity of the dimers using rat Nb2 cells, which proliferate in response to lactogens. The bioactivity of the dimers on rat Nb2 cells (Fig. 9Go) correlated well with their ability to transduce signals in human T-47D cells. This suggests that the restoration of the biological activities of hPRL-G129R and hGH-G120R was most likely due to the addition of a second functional binding site (site 1). This conclusion is further supported by our examination of truncated forms of dimeric hPRL-G129R in which a large portion of the second Site 1 (helix IV) was deleted (Fig. 10Go). It is noteworthy that three types of responses were observed in the Nb2 cell proliferation assay in response to these truncated mutants, including full or near full agonism (PRL, G129R2, and G129R2{Delta}C25), partial agonism (G129R, G129R2{Delta}C40, G129R2{Delta}C60, and G129R2{Delta}C180), and minimal agonism (G129R2{Delta}C80, G129R2{Delta}C100, G129R2{Delta}C120, G129R2{Delta}C142, and G129R2{Delta}C160). With the exception of G129R 2{Delta}C180, truncations into and beyond helix III completely abolished the partial agonism, suggesting that helix III in the second hPRL-G129R moiety is involved in the partial agonism observed in G129R2{Delta}C25, G129R2{Delta}C40,and G129R2{Delta}C60. One possibility is that the role of helix III is merely to maintain the integrity of the second hPRL-G129R moiety. Loss of helix III may prevent the formation of a helical bundle, and the remnants of the second moiety may take on a structure that sterically hinders the ability of the first hPRL-G129R moiety to interact with the PRLR. Radioreceptor binding assays were not performed for these truncated variants of dimeric hPRL-G129R because of low production yields.

In addition to studying homodimeric hPRL-G129R, we demonstrate that homodimeric hPRL binds to hPRLRs (Fig. 2Go), activates PRLR-associated signaling pathways (Fig. 7CGo), and exhibits bioactivity in vitro (Fig. 9BGo) similar to that of monomeric hPRL (Figs. 2Go, 7AGo, and 9BGo). Similar results were observed for homodimeric hGH (Figs. 8CGo and 9DGo) when compared with monomeric hGH (Figs. 8AGo and 9DGo). To examine whether the pharmacokinetic behavior of homodimers differs from that of monomers, we injected mice with monomeric and homodimeric hPRL and measured the plasma concentrations over time. We demonstrate that homodimeric hPRL has twice the area under the plasma concentation vs. time curve as monomeric hPRL (data not shown), suggesting that the larger size of the ligand enhances its pharmacokinetic behavior. This suggests that homodimers of ligands in this cytokine family could be more clinically efficacious. The presence of four potential binding sites, and the necessity for only two sites, suggests that chemical modification to further improve their pharmacokinetics may be possible. Chemical modifications such as polyethylene glycol, dextran chains, or fusion to albumin or albumin-binding peptides can affect the size and ability of a ligand to bind to a receptor; use of homodimers of a ligand for derivatization may decrease the probability of affecting crucial receptor-binding sites. Conversely, it is clear that homodimerization of antagonists would produce the opposite of what was intended. The findings in this study also suggest the possibility that the different degree of partial agonism reported by different laboratories for hPRL-G129R and other antagonists may be reflective of the number of dimeric or larger aggregates in the preparation.

Studies of the erythropoietin (EPO) have yielded results similar to those of our study. An EPO receptor antagonist (EPO-R103A) was shown to regain its biological activity when homodimerized (56). Similarly, dimeric forms of EPO have been shown be fully active and to have beneficial pharmacokinetic behavior different from that of monomeric EPO (57, 58). These findings suggest that this class of hematopoietin receptors exhibits considerable plasticity and that modifications to their cognate ligands may have therapeutic value.

In summary, we demonstrate that the ligand-binding sites of the PRLR are capable of interacting with larger molecules to induce receptor dimerization. We confirm that larger ligands with at least two functional binding sites can act as agonists and that a second receptor-binding site 1 can replace the need for a receptor-binding site 2. The recent implications of autocrine hPRL and hGH in cancers and the overexpression of their cognate receptors in certain cancers make the PRLR and GHR targets of interest for the development of therapeutics. The results of this study suggest the feasibility of designing novel multifunctional therapeutics that target the PRL/GH receptor family as we have demonstrated using hPRL-G129R fused with other proteins (G129R-IL2, G129R-Endostatin, and G129R-PE40-KDEL) (59, 60, 61).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Lines and Reagents
T-47D human breast cancer cells and transformed HEK cells (HEK 293) were obtained from the American Type Culture Collection (Manassas, VA). Nb2–11 rat pre-T lymphoma cells (Nb2) were kindly provided by Dr. Arthur Buckley. All reagents were purchased from Invitrogen (Carlsbad, CA) unless otherwise stated. T-47D cells were maintained in RPMI Medium 1640 supplemented with 10% fetal bovine serum (FBS) and 10 µg/ml gentamicin. HEK 293 cells were maintained in DMEM containing high glucose, 1 mM sodium pyruvate, 1 mM pyridoxine hydrochloride, and 2 mM L-glutamine; the medium was further supplemented with 10% FBS, 100 U/ml penicillin, and 0.7 µM streptomycin. Nb2 cells were maintained in MEM supplemented with 10% FBS, 10% horse serum, 0.1 mM nonessential amino acids, 50 µg/ml streptomycin, 50 U/ml penicillin, and 0.1 mM 2-mercaptoethanol. The cell lines were incubated at 37 C with humidity and 5% CO2.

Construction of hPRL and hPRL-G129R Homo- and Heterodimer Expression Vectors
Previously, hPRL and hPRL-G129R were cloned by inserting their cDNAs between the NdeI and XhoI sites of the expression vector pET22b (Novagen; Madison, WI). These plasmids were used as DNA templates for PCR amplification of each moiety of the recombinant dimers. The first moiety of hPRL or hPRL-G129R was amplified using the oligonucleotide primers 5'-TCATATGTTGCCCATCTGTCCCGGCG-3' and 5'-TGGATCCGCAGTTGTTGTTGTGGATG-3', which incorporate an NdeI site at the amino terminus and replace the carboxy-terminal stop codon with a BamHI site. The second moiety of hPRL or hPRL-G129R was amplified using the primers 5'-TGGATCCTTGCCCATCTGTCCCGGCG-3' and 5'-TCTCGAGTTAGCAGTTGTTGTTGTGG-3', which incorporate a BamHI site at the amino terminus and an XhoI site after the stop codon. The PCR products were ligated with pCR2.1 T/A cloning vector and transformed into E. coli TOP10 cells obtained from Invitrogen. Positive clones were identified by plating the cells on Luria-Bertani agar plates containing 100 µg/ml ampicillin and 80 µg/ml 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside. The plasmids were isolated using the QIAprep Spin Miniprep Kit (QIAGEN, Valencia, CA) and the NdeI-PRL-BamHI, NdeI-G129R-BamHI, BamH I-PRL-stop-XhoI and BamH I-G129R-stop-XhoI DNA fragments were isolated by restriction digestion and separated by electrophoresis. The DNA fragments were purified from the agarose gel using the QIAquick Gel Extraction Kit (QIAGEN) and ligated with NdeI and XhoI cut pET22b to create pET22b-PRL-PRL, pET22b-G129R-G129R, pET22b-PRL-G129R, and pET22b-G129R-PRL. A similar strategy was used to create pET22b-GH-GH and pET22b-G120R-G120R. All oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA), restriction and modifying enzymes were purchased from New England BioLabs (Beverly, MA), and PCRs were performed using the Advantage-HF Kit (BD Biosciences, Palo Alto, CA).

Production and Purification of hPRL and hPRL-G129R Homo- and Heterodimers
Chemically competent Rosetta (DE3) pLysS E. coli cells (Novagen) were transformed with the constructs and propagated overnight at 37 C with agitation in Luria-Bertani broth containing 100 µg/ml ampicillin. The next morning 40 ml of each of the starter cultures was used to inoculate 1 liter of Terrific Broth in baffled flasks, and the cultures were grown to an OD600 of 0.9 before induction of protein production with 1 mM isopropyl-ß-thiogalactoside (Alexis Biochemicals, San Diego, CA). After 4 h of induction the cells were harvested by centrifugation at 5000 x g for 5 min and resuspended in solution 1 (20 mM Tris·HCl; 10 mM EDTA; 0.5% Triton X-100; pH adjusted to 8.0) to which 0.4 mg/ml lysozyme was added. The cells were incubated at room temperature for 1 h to weaken the cell membranes before the cells were sheared on ice with five 1-min ultrasonic pulses (30% duty at setting 7) using a 550 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA). The insoluble fraction containing the inclusion bodies was recovered by centrifugation at 12,000 x g for 30 min at 4 C and resuspended in solution 2 (20 mM Tris·HCl; 10 mM EDTA; 1 M urea; pH adjusted to 8.0). The inclusion bodies were collected by centrifugation at 12,000 x g for 30 min at 4 C and were solubilized and denatured in solution 3 (20 mM Tris··HCl; 10 mM EDTA; 8 M urea; 1% vol/vol ß-mercaptoethanol; pH adjusted to 8.0). The proteins were refolded by dialysis against baths of 20 mM Tris·HCl; 10 mM EDTA, pH 8.0 containing decreasing amounts of urea (4 M urea, 2 M urea, 0 M urea, 0 M urea). The refolded proteins were filtered through 0.45 µm filters and purified by anion-exchange chromatography using a 5 ml HiTrap Q Sepharose HP column (Amersham Biosciences, Piscataway, NJ) on an ÄKTA fast protein liquid chromatography system (Amersham Biosciences). The concentration of the purified proteins was determined using the Coomassie Plus Protein Assay Reagent (Pierce Chemical Co., Rockford, IL). The purified proteins were separated by nonreducing and reducing SDS-PAGE along with BSA standards (Pierce) and stained with SYPRO Orange (Molecular Probes, Eugene, OR) to confirm their concentration and purity. The identity of the proteins was confirmed by Western blotting.

Western Blot Analysis
Purified monomers, homodimers, and heterodimers of hPRL and hPRL-G129R were separated on a 12% polyacrylamide gel by reducing SDS-PAGE. The proteins were transferred by Western blot to a Hybond enhanced chemiluminescence nitrocellulose membrane (Amersham Biosciences) at 25 V overnight. The nitrocellulose membrane was blocked for 1 h at room temperature in Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBS-T) and 5% milk. The membrane was incubated at room temperature with agitation for 4 h in the blocking solution containing a 1:5000 dilution of rabbit anti-hPRL antiserum (National Hormone and Pituitary Program, NIH, Bethesda, MD). The blots were washed three times with TBS-T and incubated for 2 h at room temperature with agitation in blocking solution containing a 1:2000 dilution of goat antirabbit-conjugated horseradish peroxidase (HRP) (Bio-Rad Laboratories, Inc., Hercules, CA). Blots were washed three times with TBS-T and incubated for 1 min with enhanced chemiluminescence Western Blotting Detection Reagents (Amersham Biosciences). The blots were exposed to Kodak Biomax MR film (Fisher Scientific), and the film was developed with a Konica SRX-101A processor (Konica Minolta Medical Imagining, Wayne, NJ).

Radioreceptor Binding Assay
One million cells per well of T-47D human breast cancer cells were seeded in six-well tissue culture plates and grown to confluency. Cells were starved in serum-free RPMI 1640 for 1 h and incubated for 2 h at room temperature in serum-free medium containing 5 x 104 cpm of 125I-labeled hPRL (specific activity, 40 µCI/µg; NEN PerkinElmer, Boston, MA) with or without various concentrations of monomeric or homodimeric hPRL and hPRL-G129R. An excess of hPRL was used to determine nonspecific binding. Cells were washed three times with serum-free RPMI 1640 and lysed in 0.5 ml of 0.1 N NaOH/1% sodium dodecyl sulfate. Bound radioactivity was determined using a Beckman LS6500 Liquid Scintillation Counter (Beckman Coulter, Inc., Fullerton, CA) calibrated to detect 125I using a wide spectrometer window. The percentage of specific binding was calculated and compared among the samples.

BRET2 Assay
BRET2 assays were performed precisely as described by Tan et al. (53). Briefly, HEK 293 cells were seeded in six-well tissue culture plates at a density of 5 x 105 cells per well and grown overnight to 90–95% confluency. Eukaryotic expression vectors encoding the PRLR tagged with GFP2 (LF-GFP2, SF1a-GFP2, SF1b-GFP2) and Rluc (LF-Rluc, SF1a-Rluc, SF1b-Rluc) at the carboxy terminus were transiently transfected into the cells. After 48 h incubation the transfected cells were washed, detached, and resuspended in BRET2 buffer at a density of 2 x 106 cells per ml. After incubation at 37 C for at least 1 h, the relative expression levels of the GFP2- and Rluc-tagged PRLR constructs were estimated by comparing their fluorescence and bioluminescence to that of cells transfected with a GFP2-Rluc fusion plasmid. Bioluminescence scanning and BRET2 signal detection were carried out immediately after the addition of the ligand (45.7 nM) and 5 µM of DeepBlueC. The bioluminescence from the oxidation of the DeepBlueC by Rluc has a peak emission at 390–405 nm, and the fluorescence from the GFP2 acceptor has a peak emission at 500–520 nm. Energy transfer from Rluc to GFP2 was defined as the BRET ratio, which was determined by calculating the ratio of light emitted by receptor-GFP2 (500–520 nm) over the light emitted by receptor-Rluc (385–420 nm). Values were corrected by subtracting the BRET background signal detected in nontransfected cells (Emission500-520 nm – background500-520 nm)/(Emission385-420 nm – background385-420 nm).

STAT5, Erk1/2, and Akt Phosphorylation Assays
T-47D cells were resuspended in RPMI 1640 supplemented with 10% charcoal/dextran treated FBS (HyClone Laboratories, Inc., Logan, UT) and 10 µg/ml gentamycin and plated in 12-well tissue culture plates. The cells were grown overnight to approximately 80% confluency and depleted for 1–72 h in RPMI 1640 supplemented with 0.5% charcoal/dextran-treated FBS to determine which condition best achieved the lowest background activation of signaling molecules. Because prolonged depletion produced no improvement in this regard, 1-h depeletions were used. Cells were treated for the indicated amounts of times with the indicated concentrations of monomeric, homodimeric, or heterodimeric hPRL, hPRL-G129R, hGH, or hGH-G120R. The cells were washed with ice-cold PBS and lysed in 100 µl of Lysis Buffer (20 mM Tris··HCl, pH 7.4; 1% Igepal CA630; 6 mM deoxycholic acid; 150 mM NaCl; 1 mM EDTA, pH 8.0) containing protease inhibitors (1 µg/ml aprotinin; 1 µg/ml leupeptin; 170 µg/ml phenylmethylsulfonylfluoride; 180 µg/ml sodium orthovanadate). The lysates were homogenized and incubated on ice for 20 min before clarification by centrifugation at 12,000 x g at 4 C for 30 min. Approximately 40 µg of the supernatants were used for Western blot analysis as described above using 10% or 4–15% polyacrylamide gels.

Blots were initially performed with a 1:1000 dilution of mouse anti-phospho STAT5A/B antibody (Upstate Biotechnology, Inc., Lake Placid, NY) and a 1:2000 dilution of goat antimouse-HRP conjugate (Bio-Rad). Blots were reprobed with a 1:1333 dilution of rabbit anti-STAT5 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and a 1:2000 dilution of goat antirabbit HRP conjugate (Bio-Rad); a 1:1333 dilution of mouse anti-phospho ERK (Santa Cruz Biotechnology), and a 1:2000 dilution of goat antimouse-HRP conjugate; and lastly with a 1:200 dilution of rabbit anti-phospho Akt antibody (Santa Cruz Biotechnology) and a 1:2000 dilution of goat antirabbit HRP conjugate.

Rat Nb2 Cell Proliferation Assay
Rat Nb2 cells were resuspended to a final concentration of 5.5 x 105 cells per ml in MEM supplemented with 0.1 mM nonessential amino acids, 10% low lactogen horse serum (catalog no. 2921154; MP Biomedicals, Irvine, CA), 50 µg/ml streptomycin, 50 U/ml penicillin, and 0.1 mM 2-mercaptoethanol. The cells were depleted overnight and reseeded in flat bottom 96-well plates at a density of 20,000 cells per well in 150 µl of medium. Treatments were added to each well in 50 µl volumes, and the cells were incubated for 72 h. The relative number of cells was determined by incubating the cells for 4 h with 40 µl of MTS/phenazine methosulfate (MTS/PMS) solution (CellTiter 96 AQueous non-radioactive cell proliferation kit; Promega, Madison, WI) per well. The reduction of MTS by living cells was determined by measuring the absorbance at 490 nm using a Benchmark microplate reader (Bio-Rad). Preliminary experiments demonstrated that cell plating densities up to 45,000 showed a linear relationship to OD 490. All assays were performed at least three times in quadruplicate.


   ACKNOWLEDGMENTS
 
We thank Ryan Westberry, Lori Holle, Michele Scotti, and Blake Derrick for their contributions to this project and Seth Tomblyn and Susan Peirce for their critical reading of the manuscript. Special thanks to Cynthia Chen, whose play on words appears in the title.


   FOOTNOTES
 
This work was supported in part by the Endowment Fund of the Greenville Hospital System, National Institutes of Health (NIH) Grant CA105479 (to W.Y.C.), and the Susan G. Komen Foundation Grant BCTR-0402985 (to W.Y.C.). Work in the Walker laboratory was supported by NIH Grant DK61005.

J.F.L., D.T., A.M.W. and W.Y.C. have nothing to declare related to this study.

First Published Online November 3, 2005

Abbreviations: BRET, Bioluminescence resonance energy transfer; ECD, extracellular domain; EPO, erythropoietin; FBS, fetal bovine serum; GFP2, variant of green fluorescent protein; GHR, GH receptor; HEK, human embryonic kidney; hGH-G120R, hGHR antagonist; hPRL-G129R, hPRLR antagonist; HRP, horseradish peroxidase; LF, long form of the human PRLR; mAb, monoclonal antibody; MEK, MAPK kinase; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PL, placental lactogen; PRL, prolactin; PRLR, PRL receptor; Rluc, Renilla reniformis luciferase; SF1a, short form 1a of the human PRLR; SF1b, short form 1b of the human PRLR; STAT5, signal transducer and activators of transcription 5; TBS, Tris-buffered saline; TBS-T, TBS containing 0.05% Tween 20.

Received for publication September 6, 2005. Accepted for publication October 28, 2005.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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